The present invention relates to modified and variant forms of Interleukin-5 molecules capable of antagonizing or reducing the activity of IL-5 and their use in ameliorating, abating or otherwise reducing the aberrant effects caused by native or mutant forms of IL-5.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the description.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly important in the area of haemopoietic growth factor research where a number of disease conditions are predicated on the aberrant effects of native or mutant forms of growth factors.
One particularly important haemopoietic growth factor is IL-5. This molecule is a lymphokine secreted by T-cells and mast cells and is a disulfide-linked homodimeric glycoprotein. The human form of this molecule comprises 114 amino acids per monomer. IL-5 consists of a bundle of four a-helices in an up-up, down-down array. The phenomenon of D-helix swapping whereby one bundle is built up of three helices coming from one monomer and a fourth helix which is contributed by the second monomer is unique to IL-5. The IL-5 molecule also contains two short anti-parallel xcex2-strands located between helices A and B and helices C and D.
Human and murine IL-5 receptors comprise two different chains, the xcex1 and xcex2-subunits.
Human IL-5 binds to the xcex1-subunit but the binding affinity is increased upon association with the xcex2-chain. The xcex2-chain is shared by other cytokines such as GM-CSF and IL-3.
IL-5 is a haemopoietic growth factor with selectivity for production and activation of human eosinophils. There is a need, therefore, to develop antagonists of IL-5 to act as therapeutic agents for chronic asthma or other disease states with demonstrated eosinophilia or other conditions associated with IL-5. It is also important for the IL-5 antagonist not to interfere with the activities of other cytokines, such as GM-CSF or IL-3.
Accordingly, one aspect of the present invention contemplates a modified IL-5 comprising a sequence of amino acids within a first a-helix wherein one or more exposed amino acids in said first a-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or a non-acidic amino acid residue.
The IL-5 which is subject to modification is generally of mammalian origin such as from humans, primates, livestock animals (eg. sheep, cows, pigs, horses), laboratory test animals (eg. mice, rats, guinea pigs, rabbits), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, foxes, deer). Most preferably, the IL-5 is of human origin. The modified IL-5 of the present invention may be glycosylated or unglycosylated and does not interfere with GM-CSF or IL-3 activity.
Even more particularly, the present invention is directed to a modified human IL-5 molecule comprising a sequence of amino acids wherein Glu at amino acid position 13 (or its equivalent position) in a first a-helix is replaced by Arg or Lys or a chemical equivalent or derivative thereof. An alternative substitution may also be made using non-acidic amino acid residues such as but not limited to Gln and Asn or their chemical equivalent or derivatives. A xe2x80x9cderivativexe2x80x9d may be a naturally occurring or synthetic amino acid residue.
In accordance with the present invention, it is proposed that the modified IL-5 molecules defined above act as antagonists of the native form of IL-5. The term xe2x80x9cmodifiedxe2x80x9d is considered herein synonymous with terms such as xe2x80x9cvariantxe2x80x9d, xe2x80x9cderivativexe2x80x9d and xe2x80x9cmutantxe2x80x9d. The present invention is particularly directed to a modified IL-5 which exhibits specific antagonism of IL-5 mitogenic effects such as observed in vitro. The modified IL-5 molecules may be glycosylated or unglycosylated and do not interfere with GM-CSF or IL-3 activity.
Accordingly, another aspect of the present invention is directed to an IL-5 antagonist said antagonist comprising an IL-5 molecule having an amino acid sequence in its first at-helix wherein one or more exposed amino acids in said first xcex1-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or a non-acidic amino acid residue.
More particularly, the present invention provides an antagonist of IL-5 said antagonist comprising an IL-5 molecule with Gln at position 13 (or its equivalent position) in a first xcex1-helix substituted by Arg or Lys or a chemical equivalent or derivative thereof An alternatively substitution may also be made using non-acidic amino acid residues such as but not limited to Gln and Asn or their chemical equivalents or derivatives.
The modified IL-5 molecule of the present invention is preferably in recombinant or synthetic form and, with the exception of the amino acid substitution(s) in the first a-helix, the amino acid sequence of the IL-5 may be the same as the naturally occurring molecule (i.e. native molecule) or may carry single or multiple amino acid substitutions, deletions and/or additions to the native amino acid sequence. It is then referred to as a xe2x80x9cmutantxe2x80x9d IL-5. The structure of the first xcex1-helix of IL-5 has been determined at 2.4 angstrom resolution by X-ray crystallography and comprises amino acid residues 7 to 27 or their equivalents (see Milburn et al. Nature 363: 172-176, 1993). The modified IL-5 of the present invention may or may not comprise a leader sequence.
The nucleotide and corresponding amino acid sequence for the modified IL-5 having Arg in position 13 is shown in SEQ ID NOs: 1 and 2 and FIG. 1. The leader sequence is shown as amino acids 1 to 6 (Met His Tyr His His His [SEQ ID NO:3]). Consequently, amino acids 7 to 27 are shown as amino acids 13-33 in SEQ ID NOs: 1 and 2 and in FIG. 1. Reference to amino acids 7 to 27 is taken as amino acid residue numbers in a molecule without a leader sequence. The amino acid sequence for amino acids 7 to 27 is shown as SEQ ID NO:4 except that amino acid 13 is represented as Xaa. In accordance with the present invention Xaa is preferably a basic amino acid residue or a non-acidic amino acid residue.
Reference to xe2x80x9cunglycosylated formxe2x80x9d herein means that the molecule is completely unglycosylated such as when expressed in recombinant form in a prokaryotic organism (e.g. E. coli). Alternatively, a glycosylation-deficient mammalian cell may be used or complete deglycosylation may occur in vitro using appropriate enzymes. Different glycosylation patterns are encompassed by the present invention such as when recombinant molecules are produced in different mammalian cells.
An xe2x80x9cexposedxe2x80x9d amino acid is taken herein to refer to an amino acid on a solvent-exposed or outer portion of an xcex1-helix compared to those amino acids orientated towards the inside of the molecule.
An acidic amino acid includes, for example, Glu and Asp. Preferred basic amino acids are Arg and Lys. Preferred non-acidic amino acids are Gln and Asn.
According to another aspect of the present invention there is provided a modified IL-5 characterised by:
(i) comprising a sequence of amino acids within a first xcex1-helix,
(ii) having one or more exposed amino acids in said xcex1-helix which have acidic or acidic-like properties being substituted by a basic amino acid residue or a non-acidic amino acid residue;
(iii) being in recombinant or synthetic form; and
(iv) being capable of antagonising IL-5 mitogenic activity in vitro.
In a related embodiment, the present invention provides an IL-5 antagonist characterised by:
(i) comprising a sequence of amino acids within a first xcex1-helix;
(ii) having one or more exposed amino acids in said xcex1-helix which have acidic or acidic-like properties being substituted by a basic amino acid residue or a non-acidic amino acid residue;
(iii) being in recombinant or synthetic form; and
(iv) being capable of antagonising IL-5 mitogenic activity in vitro.
The IL-5 mutant may be in glycosylated or unglycosylated form.
This aspect of the present invention is predicated in part on the finding that a mutation in amino acid 13 (Glu) of human (h) IL-5 to Arg results in the IL-5 variant being capable of antagonising IL-5 mitogenic effects in vitro. This variant is referred to herein as xe2x80x9cIL-5 Arg13xe2x80x9d xe2x80x9cE13 Rxe2x80x9d. Such a variant would be unable to bind to high affinity receptors but would still able to fully bind the low affinity a chain of the IL-5 receptor. Importantly, the IL-5 Arg13 mutant acts as an antagonist, preventing the stimulatory effect of native IL-5. A particularly important use of the IL-5 Arg13 antagonist is in reducing or otherwise antagonising IL-5-mediated stimulation and activation of eosinophils in vivo or in vitro. The antagonist may also be able to antagonise effects caused by a mutated, endogenous IL-5. The present invention extends to a range of other IL-5 mutants such as IL-5 Lys13, IL-5 Gin13 and IL-5 Asn13 as well as functionally equivalent mutants. The nucleotide and corresponding amino acid sequence for IL-5 Arg13 (E13R) are shown in SEQ ID NOs: 1 and 2. The present invention extends, in a particularly preferred embodiment, to an isolated polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO:2 or a genetic sequence encoding same having a nucleotide sequence substantially as set forth in SEQ ID NO:1.
By way of a shorthand notation, both single and three letter abbreviations are used for amino acid residues in the subject specification and these are defined in Table 1.
Where a specific amino residue is referred to by its position in IL-5, a single or three letter amino acid abbreviation is used with the residue number given in superscript (eg. wherein Xaan, wherein Xaa is the amino acid residue) and xe2x80x9cnxe2x80x9d is the residue number in the molecule.
The present invention is exemplified using IL-5 Arg13. This is done, however, with the understanding that the present invention extends to all other IL-5 variants having antagonistic properties to IL-5 in vitro or against eosinophils in vitro or in vivo.
According to another aspect of the present invention there is provided an IL-5 variant comprising an amino acid sequence in the first xcex1-helix of said IL-5:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu lie Ala [SEQ ID No. 4];
wherein Xaa is a basic or non-acidic amino acid residue preferably selected from the group consisting of Arg, Lys, Gln and Asn and wherein said variant IL-5 acts as an antagonist for at least one property of the corresponding native IL-5. The amino acid sequence defined by SEQ ID NO:4 corresponds to amino acid residues 7 to 27 of human IL-5. Preferably, Xaa is Xaan wherein n is amino acid position 13 of human IL-5. Preferably Xaan is Arg13 or its equivalent.
In a related embodiment, the present invention contemplates an IL-5 antagonist comprising a polypeptide or chemical equivalent thereof comprising amino acids 7 to 27 of the first xcex1-helix of human IL-5 with the proviso that one or more exposed amino acids in said first xcex1-helix having acidic or acidic-like properties are substituted by a basic amino acid residue or a non-acidic amino acid residue.
Preferably, the acidic or acidic-like amino acid residue if Glu or Asp and is replaced by Arg, Lys, Gln or Asn.
More preferably, the acidic or acidic-like amino acid residue is replaced by Arg.
Most preferably, the amino acid sequence of the modified IL-5 is as set forth in SEQ ID NO:2.
In a particularly preferred embodiment, the present invention is directed to an IL-5 antagonist comprising a modified IL-5 molecule having the following amino acid sequence in its first xcex1-helix:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala [SEQ ID NO:4]
wherein Xaa is Arg;
or an IL-5 molecule having a single or multiple mutation in its first xcex1-helix giving functionally similar antagonistic properties to the mutation wherein Xaa is Arg.
In respect of the latter embodiment, the mutation in the IL-5 molecule may be a single or multiple amino acid substitution, deletion and/or addition or may be an altered glycosylation pattern amongst other mutations. Preferably, the IL-5 antagonist comprises an amino acid sequence as set forth in SEQ ID NO:2.
The IL-5 antagonists of the present invention and in particular IL-5 Arg13 are useful inter alia in the treatment of allergy, some myeloid leukemias (such as eosinophilic myeloid leukaemia), idiopathic eosinophilic syndrome, allergic inflammations such as asthma, rhinitis and skin allergies by preventing or reducing IL-5-mediated activation of eosinophils. These and other conditions are considered herein to result from or be facilitated by the aberrant effects of an endogenous native IL-5 or an endogenous naturally mutated IL-5.
The present invention, therefore, contemplates a method of treatment comprising the administration to a mammal of an effective amount of a modified IL-5 as hereinbefore defined and in particular IL-5 Arg13 for a time and under conditions sufficient for effecting said treatment.
Preferably, the treatment is in respect of the treatment of allergy, some myeloid leukemias (such as eosinophilic myeloid leukaemia), idiopathic eosinophilic syndrome, allergic inflammations such as asthma, rhinitis and skin allergies.
Generally, the mammal to be treated is a human, primate, livestock animal, companion animal or laboratory test animal. Most preferably, the mammal is a human.
A single modified IL-5 may be administered or a combination of variants of the same IL-5. For example, a range of IL-5 variants could be used such as a combination selected from two or more of IL-5 Arg13, IL-5 Lys13, IL-5 Gln13 and IL-5 Asn13. The IL-5 present invention are particularly useful in treating eosinophilia and conditions resulting therefrom such as asthma. Administration is preferably by intravenous administration but a range of other forms of administration are contemplated by the present invention including via an implant device or other form allowing sustained release of the IL-5 variant, in a nebuliser form or nasal spray. Modified forms of IL-5 permit entry following topical application are also encompassed by the present invention.
In addition to the modifications to IL-5 contemplated above, the present invention further provides a range of other derivatives of IL-5.
Such derivatives include fragments, parts, portions, mutants, homologues and analogues of the IL-5 polypeptide and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to IL-5 or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding IL-5. Derivatives also contemplated modifications to resident nucleotides. Alteration of the nucleotides may result in a corresponding altered amino acid sequence or altered glycosylation patterns amongst other effects. xe2x80x9cAdditionsxe2x80x9d to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to xe2x80x9cIL-5xe2x80x9d includes reference to all derivatives thereof including functional derivatives or IL-5 immunologically interactive derivatives. All such derivatives would be in addition to the modifications to the first xcex1-helix contemplated above. Accordingly, such derivatives would be to IL-5 Arg13, IL-5 Lys13, IL-5 Gln13 or IL-5 Asn13.
Analogues of IL-5 contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
The present invention further contemplates chemical analogues of IL-5 capable of acting as antagonists or agonists of IL-5 or which can act as functional analogues of IL-5. Chemical analogues may not necessarily be derived from IL-5 but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of IL-5. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.
Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a fully glycosylated molecule and from a naturally glycosylated molecule to molecules with an altered glycosylation pattern. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
All these types of modifications may be important to stabilise the modified IL-5 molecules if administered to an individual or when used as a diagnostic reagent. The modifications may also add, complement or otherwise facilitate the antagonistic properties of the modified IL-5 molecules.
Reference herein to a xe2x80x9cmodifiedxe2x80x9d IL-5, therefore, includes reference to an IL-5 with an altered amino acid sequence in the first xcex1-helix as well as, where appropriate, a range of glycosylation variants, amino acid variations in other parts of the molecule, chemical modifications to the molecule as well as fusion molecules.
The present invention also provides a pharmaceutical composition comprising the modified IL-5 molecules as hereinbefore defined or combinations thereof.
Accordingly, the present invention contemplates a pharmaceutical composition comprising a a modified IL-5, said modified IL-5 comprising a sequence of amino acids with a first xcex1-helix wherein one or more exposed amino acids in said first xcex1-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or non-acidic amino acid residue, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.
In a related embodiment, the present invention provides a pharmaceutical composition comprising a modified human IL-5 or a mammalian homologue thereof said modified IL-5 comprising a sequence of amino acids in a first xcex1-helix of:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala [SEQ ID NO:4]
wherein Xaa is a basic non-acidic amino acid residue preferably selected from Arg, Lys, Gln and Asn, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents. Preferably, Xaa is Xaan where n is amino acid position 13. Preferably, Xaa is Arg.
In another related embodiment, the present invention contemplates a pharmaceutical composition capable of antagonising IL-5, said composition comprising a modified IL-5 having an amino acid sequence in its first xcex1-helix:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala [SEQ ID NO:4]
wherein Xaa is selected from Arg, Lys, Gln and Asn.
Preferably, Xaa is Arg.
The pharmaceutical compositions may also contain other pharmaceutically active molecules including other IL-5 variants. The modified IL-5 molecule and other components in a pharmaceutical composition are referred to below as xe2x80x9cactive agentsxe2x80x9d or xe2x80x9cactive compoundsxe2x80x9d.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, mannitol glycine or suitable mixtures thereof. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. Mannitol glycine is a particularly useful formulation especially when the modified IL-5 molecules are given as an intravenous drip.
The present invention also extends to forms suitable for inhaling such as a nasal spray as well as in nebulizer form. Alternatively, sustained release compositions may be formulated as well as a range of implant devices. When suitably modified, the molecules may also be given as a cream, lotion or gel. A suitable carrier for a cream, lotion or gel includes a polyol such as but not limited to glycerol, propylene glycol, liquid polyethylene glycol and the like.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, and antibacterial and antifungal agents. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated by the present invention. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition involving or facilitated by aberration of IL-5 molecules or levels of IL-5.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 xcexcg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 xcexcg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating modified IL-5 expression or modified IL-5 activity. The vector may, for example, be a viral vector.
Still another aspect of the present invention is directed to antibodies to the modified IL-5 molecules and their derivatives. Such antibodies may be monoclonal or polyclonal and may be specifically raised to modified IL-5 or derivatives thereof. In the case of the latter, a modified IL-5 or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant modified IL-5 or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
For example, a modified IL-5 and its derivatives can be used to screen for naturally occurring antibodies to IL-5. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for a modified or mutant IL-5. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of IL-5 levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols when modified IL-5 molecules are employed.
Antibodies to modified IL-5 of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A xe2x80x9csynthetic antibodyxe2x80x9d is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.
For example, specific antibodies can be used to screen for modified IL-5 proteins. The latter would be important, for example, as a means for screening for levels of modified IL-5 in a cell extract or other biological fluid or purifying modified IL-5 made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of modified IL-5.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of modified IL-5 or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
Another aspect of the present invention contemplates a method for detecting modified IL-5 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for modified IL-5 or its derivatives or homologues for a time and under conditions sufficient for an antibody-modified IL-5 complex to form, and then detecting said complex.
The present invention also contemplates genetic assays such as involving PCR analysis to detect a modified IL-5 gene or its derivatives. Alternative methods include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphism analysis (SSCP) as well as specific oligonucleotide hybridisation.
The present invention extends to nucleic acid molecules encoding a modified IL-5 of the present invention. Such nucleic acid molecules may be DNA or RNA. When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.
Although the nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic molecules such as vector molecules and in particular expression vector molecules. Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell. Preferably, prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.
Accordingly, another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human modified IL-5 gene portion, which modified IL-5 gene portion is capable of encoding an modified IL-5 polypeptide or a functional or immunologically interactive derivative thereof
Preferably, the modified IL-5 gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said modified IL-5 gene portion in an appropriate cell.
In addition, the modified IL-5 gene portion of the genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-S-transferase or part thereof
Accordingly, another aspect of the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a modified IL-5, said modified IL-5 comprising a sequence of amino acids with a first xcex1-helix wherein one or more exposed amino acids in said first xcex1-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or a non-acidic amino acid residue.
Preferably the sequence of nucleotides encodes a human modified IL-5 or a mammalian homologue which comprises a sequence of amino acids in a first xcex1-helix of:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala [SEQ ID NO:4]
wherein Xaa is a basic or non-acidic amino acid residue preferably selected from Arg, Lys, Gln and Asn and wherein said modified IL-5 exhibits antagonism of IL-5 induced mitogenic effects. Preferably, Xaa is Xaan where n is amino acid position 13. Preferably, Xaa is Arg.
The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.
The present invention further contemplates the use of a modified IL-5 as hereinbefore defined in the manufacture of a medicament in the treatment of allergy, some myeloid leukemias (such as eosinophilic myeloid leukaemia), idiopathic eosinophilic syndrome, allergic inflammations such as asthma, rhinitis and skin allergies.
The present invention is further described by reference to the following non-limiting Examples and/or Figures.